It is well known that a sample of normal human erythrocytes does not consist of a homogenous population. The age of such cells varies from zero to 120 days, and their densities range between 1.065 to 1.095 grams (g) per milliliter (ml) with the reticulocytes and youngest erythrocytes having the lowest density. See A. F. Wu and T. T. Wu, Velocity Sedimentation in Isotonic Saline and Sucrose Buffer as a Possible Method of Fractionating Normal Erythrocytes, Preparative Biochemistry, 8:347-361 (1978), and references cited therein.
As described in the aforementioned publication, when red cells mature their density gradually increases. Most of them acquire a biconcave disc shaped form known as discocytes, while some of them display short spicules projecting from a central spheroid and are designated echinocytes. Their dimensions are also quite varied. In hypotonic solutions, erythrocytes tend to burst but not all at once. Roughly half of them will lyse in 0.40 to 0.45% saline. On storing such cells for one day, the concentration of saline required to maintain half lysis must be increased to 0.47 to 0.58%. The longer red cells are stored, the more fragile they become. Such osmotic fragility does not appear related, however, to the reticulocyte count of the blood nor to the age of the erythrocytes.
It has been customary to assume that the age of erythrocytes varies linearily with density and, therefore, prior efforts to investigate the different cell populations of erythrocyte samples have commonly utilized equilibrium sedimentation techniques. R. C. Leif and J. Vinograd, The Distribution of Buoyant Density of Human Erythrocytes in Bovine Albumin Solutions, Proc. Nat. Acad. Sci., 51:520-524 (1964). In a linear density gradient, human red cells exhibit only one peak centered around 1.08 g/ml, but in a stepwise gradient consisting of isotonic solutions with densities of 1.07, 1.08, and 1.09 g/ml and a cushion at the bottom, four bands of cell populations based on differences in buoyant density have been identified.
Velocity sedimentation in a density gradient of bovine serum albumin has also been used experimentally to investigate human red cell populations but again only one peak was obtained. N. Catsimpoolas, Methods of Cell Separation, Vol. 1 (Plenum Press, N.Y., 1977). Whether by velocity sedimentation or equilibrium sedimentation, such fractionation of red cells has been carried out only on an experimental basis for the purpose of investigating red cell populations. Indeed, the density gradient medium used in such experimental work (bovine serum albumin) has the effect of coating the cells and precludes their subsequent use in clinical procedures.
One aspect of this invention lies in the recognition that for many of those patients suffering from various hemolytic anemias and other diseases requiring transfusions of packed red cells, the presence of damaged or drying cells in the transfusate only increases the burden on the patient's system and is especially undesirable where, for example, the patient's condition is complicated by congestive heart disease, renal failure, cirrhosis, or the like. This invention is thus concerned with a simple but highly effective procedure for fractionating a heterogenous red cell population, such as that of donor blood which has been stored for a number of days awaiting transfusion, to exclude those cells which are approaching lysis and are therefore nearing the end of their useful life.
Another aspect of this invention resides in the discovery that although red cells are known to increase in density as they become older, buoyant density fractionating procedures are unsuitable for this purpose because such procedures would result in the elimination of older cells but not necessarily those nearing lysis, and that on the other hand, a velocity sedimentation procedure may be used effectively to exclude those cells approaching lysis regardless of their age. The result is a method which may be used effectively to segregate cells according to their viability, and which allows the retention for transfusion of those human red cells that are more viable regardless of their age or density.
In brief, the method of this invention lies in exposing an entire red cell population to velocity sedimentation in a solution of isotonic saline buffered to a pH of about 7.4 and containing a soluble cell-compatible density-adjusting agent in an amount sufficient to adjust the solution to a density within the range of about 1.005 to 1.015. Particularly effective results have been achieved when the density-adjusting agent is a disaccharide such as sucrose. The red cells are collected and separated into at least two fractions based on differences in sedimentation rates, and that fraction containing the slower sedimenting cells, particularly echinocytes, is excluded. The normal red cells of the retained fraction may then be washed in isotonic saline (to remove the sucrose) and then transfused.
Other features, advantages, and objects of the invention will become apparent as the specification proceeds.